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In response to salinity and drought, some higher plants accumulate the secondary metabolite glycinebetaine which functions as a compatible osmolyte (Rhodes and Hanson, 1993). Choline, a precursor of glycinebetaine (Rhodes and Hanson, 1993), is also a component of a primary metabolite phosphatidylcholine, an ubiquitous membrane phospholipid (Moore, 1982). In leaves of the glycinebetaine accumulator spinach, choline is synthesized as phosphocholine (PCho) and PCho is synthesized by three sequential N-methylations of phosphoethanolamine (PEA) ~ phosphomethyl-EA (PMEA) ~ phosphodimethyl-EA (PDEA) ~ PCho. The methyl group donor is Sadenosyl- L-methionine, SAM (Summers and Weretilnyk, 1993). The enzyme SAM: PMEA N-methyltransferase (PMEAMeT) is suggested to N-methylate PMEA ~ PDEA ~ PCho (Weretilnyk and Summers, 1992). A four-step strategy was developed for the partial purification of PMEAMeT on the basis of PMEA-dependent methylations (PMEAMeT activity) that involved the extraction of soluble leaf protein, ammonium sulfate precipitation, and column chromatography on DEAE Sepharose, Phenyl Sepharose and High Q Anion matrices. PDEA-dependent methylating activity co-purified with PMEAMeT activity which suggested PMEAMeT may N-methylate PMEA ~ PDEA ~ PCho. PMEAMeT was purified 43-fold and has specific activities of 14.7 and 18.0 nmol•min-1•mg-1 protein with PMEA and PDEA as substrates, respectively. Thin layer chromatography was used to identify the reaction products formed during the 30 minute assay incubation: with PMEA as the substrate, PDEA and PCho were detected in a ratio of 9: 1 as products; and with PDEA as the substrate, PCho was detected as the only product. PMEAMeT was estimated to have a native molecular mass of 76 kDa by HPLC gel filtration chromatography. Both PMEA and PDEA N-methylating activities have an alkaline pH optimum between 8.5 and 9.0 in 0.1 M Tris-HCl buffer. Neither activity was iii affected by the omission of Na2-EDT A from the assay. The addition of 10 mM Mg2+ to the assay inhibited PMEA and PDEA-dependent methylation by approximately 49% and 32%, respectively; whereas, the addition of 1 and 10 mM Mn2+ to the assay completely inhibited both activities. Both activities were inhibited by the reaction products S-adenosyl-Lhomocysteine by over 90% at 0.2 mM and PCho by approximately 80% at 10 mM. Of the products ofPCho hydrolysis, choline inhibited PMEA-dependent methylation by 10% at 10 mM; whereas, Pi inhibited PMEA and PDEA-dependent methylation by 38 and 19%, respectively at 10 mM. The compatible osmolyte glycinebetaine inhibited PMEA and PDEA-dependent methylation by between 20 and 30% at 140 mM; however, the inibition of PMEA-dependent methylation can be partly accounted for by the presence of cr ions in the assay. If present at these concentrations in the same subcellular compartment, these metabolites could serve as regulators ofPMEAMeT activities in vivo. Study of PMEAMeT contributed to identifying the number of enzymes that Nmethylate PEA ~ PMEA ~ PDEA ~ PCho and possible regulatory metabolites for choline biosynthesis in vivo. These data are pertinent to basic research and also to genetic-engineering studies aimed at introducing the glycinebetaine-accumulating trait into crop plants as an approach to enhancing osmotic-stress resistance. / Thesis / Master of Science (MSc)

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第22473号 / 工博第4734号 / 新制||工||1739(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 梅田 眞郷, 教授 森 泰生, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

We report on 2 cases of chronic relapsing thrombotic thrombocytopenic purpura, in which anti-phospholipid antibodies were also found. The first patient was felt to have the antiphospholipid antibody syndrome, while the second patient had anti-phospholipid antibodies without clinical manifestations of the anti-phospholipid antibody syndrome. We discuss chronic relapsing thrombotic thrombocytopenic purpura and the anti-phospholipid antibody syndrome. Furthermore, we introduce the possibility of an association between chronic relapsing thrombotic thrombocytopenic purpura and the presence of anti-phospholipid antibodies.

Calcium/phospholipid-dependent protein kinase (PKC) was partially purified from P1798 lymphosarcoma. Phospholipid-dependence was specific for phosphatidylserine. PKC phosphorylated Histone 1, with an apparent K_m of 14.1 μM. Chlorpromazine, a lipid-binding drug, inhibited PKC activity by 100%. Further studies were undertaken to establish analytical conditions which could be applied to the study of PKC in intact cells. The conditions included (1) determining optimum cell concentration for measuring PKC activity, (2) recovering PKC into the soluble fraction of cell extracts, (3) evaluating calcium and phospholipid requirements of PKC in this fraction, and (4) inhibiting PKC in this fraction. Final studies involved treatment of intact cells with potential activators. Both phytohaemagglutinin and a phorbol ester increased PKC activation.